1. Field of the Invention
The present invention relates to attaching a phosphorous dendrimer to magnetic microparticles.
2. Description of the Prior Art
The methods to fabricate magnetically responsive particles of sizes ranging from a few nanometers to several microns have been in continuous development for more than a decade. Magnetic microparticles of various sizes, compositions and different surface functionalities are commercially available for biological separations, immunoassays, and medical imaging. In general, these particles consist of a magnetizable core (for example iron oxide) coated with a polymer onto which a reactive functional group is linked. The reactive functional group can be an epoxy, amide, carboxyl, amino, hydroxyl, among others, and it is incorporated into the magnetic microparticles during the polymerization step. The presence of these reactive functional groups allows covalent or non-covalent interaction with biological moieties such as cells, cell organelles, nucleic acids, proteins and antibodies that facilitate their separation from other organelles, organisms or substances contained in the same matrix. Since all these processes are carried out in aqueous phases, the commercially available functionalized magnetic microparticles are usually sold as a suspension in water based storage solution, which limits their use in water based reactions.
In many circumstances, such as for the specific capture of genomic nucleic acids, it is necessary to attach a particular type of linker on the magnetic microparticle before linking a capture moiety to obtain the desired performance. If the linker required is only soluble in solvents with poor water solubility, the availability of magnetic microparticles in water based solutions represents a problem for the functionalization. Furthermore, none of the commercial providers have any information pertinent to the use of the magnetic microparticles in organic solvents nor have they developed protocols for their use under these conditions.
Phosphorous dendrimers are highly branched molecules soluble in solvents such as chloroform, tetrahydrofuran, dichloromethane, and dioxane. These molecules possess many characteristics that can enhance and facilitate capture of genomic nucleic acids in a single step when used as linkers in magnetic microparticles and other materials. A method to covalently attach a phosphorous dendrimer onto magnetic microparticles with primary amine functionality (NH2) was previously disclosed (US Patent Publication 2008/0293594, the entire contents of which are incorporated herein by reference). Since the magnetic microparticles are suspended in water and the phosphorous dendrimer is only soluble in dichloromethane, it was necessary to perform a solvent exchange from water to dichloromethane of magnetic microparticles. The solvent exchange was done using ethanol as a transition solvent. Previously, a single transition step was used and the functionalization was performed in a small scale (500 μg of bead aliquots). The method of the present invention optimizes this approach by incorporating additional transition steps to make it suitable for small or large scale functionalization and in a shorter period of time. Both approaches are schematically shown in FIG. 1.
The only known approach that has considered the use of a dendrimer on the surface of a magnetic microparticle is that of Pan et al. (Pan et al., “Dendrimer modified magnetite nanoparticles for protein immobilization,” Journal of Colloid and Interface Science, 284, 1-6 (2005)). However, Pan uses a cascading polyamidoamine dendrimer (PAMAM) and not a phosphorous dendrimer. Additionally, the dendrimer was synthesized on the surface of the magnetic microparticle by a stepwise deposition process through which layers of two different materials were incorporated until the desired dendrimer generations was reached. The resulting surface is covered with amine functionalities that cannot withstand the conditions required in the single step subtraction of genomic targets.